Cancer is the second leading cause of death in the United States. Alkylating agents are widely used in cancer therapy to cause DNA damage and kill the cells within the tumor. The damage caused by alkylating agents is commonly repaired by base excision repair (BER). Polymerase beta (Pol beta) is a key protein required for BER. Our laboratory has identified mutations in Pol beta in 25-30% of tumors examined. Some of these mutations result in abnormal BER function and cause uncontrolled cell growth, a common phenotype of cancerous cells. If the BER pathway cannot repair the damage, the homology-dependent DNA repair (HDR) pathway is activated to resolve the lesion. Proper HDR function is dependent upon several proteins including BRCA2 and RAD51. Cells that do not have BRCA2 or RAD51 are incapable of repairing damage and become more sensitive to DNA damaging agents. In addition, recent studies have shown that when BER is also impaired in cells that lack BRCA2, cells are even more sensitive to DNA damaging agents. However, only a subset of cancers is BRCA2-deficient and no studies have used cells lacking BER function even though there are mutations in BER proteins found in tumors. The long-term goal of the proposed studies is to test whether the downregulation of HDR in cells containing a BER-deficient cancer protein will lead to enhanced sensitivity in response to drug treatments. In this proposal, we will disable HDR by expressing a novel protein, PALB2, that interacts with key HDR proteins rendering cells more susceptible to DNA damage. We aim to test the effect of PALB2 expression in cells that harbor a BER- deficient Pol beta mutation that was originally identified in tumors. Using these tools, we will test whether alkylating agents, such as the chemotherapeutic drug temozolimide, will cause increase cell death when both BER and HDR are impaired. Our specific aims are to: 1) to test the hypothesis that compromising multiple DNA repair pathways by expressing a novel HDR protein will sensitize cells to DNA damage;2) to test the hypothesis that the inhibition of HDR in concert with the expression of a cancer-associated Pol beta variant will result in increased efficacy to chemotherapeutic agents;and 3) to test the hypothesis that inhibiting HDR in tumors carrying a polymerase-dead Pol beta cancer variant in vivo results in increased survival and decreased tumor growth in response to temozolimide. Targeting multiple DNA repair pathways has become an attractive strategy for cancer therapy. These studies will determine if there are therapeutic advantages for inhibiting HDR in cancers that harbor mutations in BER. The ultimate goals of this proposal are to improve the efficacy of cancer therapy while limiting the toxicity to normal tissues. The proposed studies will provide us with the potential to develop new therapeutic strategies to improve cancer survival. PUBLIC HEALTH RELEVANCE: Cells have acquired several ways to repair their DNA and many chemotherapeutic drugs deliberately damage the DNA to kill the tumor cell. It has been shown that tumor cells are frequently deficient in at least one DNA repair pathway and this deficiency leads to the increased killing of tumor cells compared to healthy cells following cancer treatments. We aim to show that expressing a novel combination of DNA repair proteins, we can shut down DNA repair pathway which has the potential to lead to the creation of new drugs to treat patients with cancers deficient in DNA repair.